Project Summary/Abstract The goal of this project is to understand the function of phosphorylation of the long isoform of TIN2 and its relation to pathogenic variants causing dyskeratosis congenita (DC). DC is an inherited bone marrow failure syndrome caused by disruption of telomere maintenance and function. Patients are at risk of life-threatening myelodysplastic syndrome, immunodeficiency, various cancers, pulmonary fibrosis, and liver disease. Hematopoietic cell transplantation addresses low blood counts, yet a better understanding will be required to treat other clinical features and the underlying cause. Monoallelic pathogenic variants in TINF2 (encodes TIN2) account for approximately 11% of DC-spectrum disorders. Previous studies have focused on the shorter of two TIN2 isoforms, and the mechanism by which pathogenic variants in TINF2 cause DC remains unclear. TIN2 interacts with TRF2, a shelterin component that directly binds telomeric DNA. Multiple DNA processing factors are targeted to telomeres through interaction with TRF2. These factors are involved in regulating the length of the telomeric single stranded 3? overhang or are involved in telomere homologous recombination. Single stranded 3? overhang DNA is required for homologous recombination and excess telomere homologous recombination can result in massive telomere shortening. Preliminary data leads to the hypothesis that (1) dynamic phosphorylation of TINL regulates competition of TIN2L and processing factors for interaction with TRF2 and localization to telomeres by altering TIN2L conformation and (2) TIN2L altered phosphorylation or the DC-associated R282H disrupts this regulation, resulting in telomere loss via hyperresection and homologous recombination. To test this hypothesis, this study first proposes to knock-in a tag to the C-terminus of endogenous TIN2L. Tagged TIN2L will be monitored for cell-cycle specific phosphorylation and recruitment to TRF2 and telomeres. The correlation of processing factor recruitment to TRF2 and telomeres throughout the cell-cycle will be assessed. Next, it proposes to knock-in phosphodead and phosphomimetic mutations and, in a separate cell line, to knock-out TIN2L and integrate inducible TIN2L wild type or TIN2L R282H, the most common TIN2 DC variant. These cell lines will be used to evaluate the effect of phosphorylation and R282H on the association of TIN2L and processing factors with TRF2 and telomeres. Biochemical approaches will be used to assess the competition of TIN2L with processing factors for TRF2 binding, and to determine if phosphodead, phosphomimetic, or R282H mutations alter the conformation of TIN2L. Finally, cell and molecular biological approaches using the above cell lines will be used to determine whether these mutations lead to telomere dysfunction. Together, these proposed studies will provide insight into the role of TIN2L in telomere biology, and will help us achieve the long term goal of improving the understanding and treatment of DC and telomere biology disorders.